• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
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  • 优先权
    • 专利摘要:
    Disinfectant composition. The present invention fits in the sector of the food industry, in the sanitary field or can even be used at the domestic level. Specifically, the present invention refers to a disinfectant composition, without toxic bactericidal agents or contaminants of the environment, applicable in any type of utensil or surface exposed to bacterial contamination. The composition of the invention can also be used for the treatment of patients suffering from bacterial infections and/or intoxications caused by heavy metals. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2717798A1
    申请号:ES201731462
    申请日:2017-12-22
    公开日:2019-06-25
    发明作者:Hayani Hikmate Abriouel；El Bakali Nabil Benomar；Lerma Leyre Lavilla
    申请人:Universidad de Jaen；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] FIELD OF THE INVENTION
    [0005]
    [0006] The present invention falls within the sector of the food industry, in the healthcare field or can even be used domestically. Specifically, the present invention refers to a disinfectant composition, without toxic bactericidal agents or environmental contaminants, applicable in any type of utensil or surface exposed to bacterial contamination. The composition of the invention can also be used for the treatment of patients suffering from bacterial infections and / or patients with heavy metal poisoning.
    [0007]
    [0008] STATE OF THE TECHNIQUE
    [0009]
    [0010] A biofilm, biofilm, bacterial tapestry or microbial mat is an organized microbial ecosystem, consisting of one or several microorganisms associated with a living or inert surface, with functional characteristics and complex structures. This type of microbial conformation occurs when cells adhere to a surface or substrate, forming a community, which is characterized by the excretion of a protective adhesive extracellular matrix.
    [0011]
    [0012] A biofilm can contain approximately 15% cells and 85% extracellular matrix. This matrix is usually formed of exopolysaccharides, which form channels through which water, enzymes, nutrients and waste circulate. There the cells establish relationships and dependencies: they live, cooperate and communicate through chemical signals (quorum perception), which regulate the expression of genes differently in different parts of the community, such as a tissue in a multicellular organism.
    [0013]
    [0014] It has been found that more than 60% of bacterial infections are caused by biofilms. For this reason, they have been widely studied and are considered a blunt clinical threat as they are capable of growing in catheters and medical and surgical utensils. In the health field it is where there is more susceptibility to nosocomial infections.
    [0015] In addition to the clinical or health field, it is important to take into account the formation of biofilms on surfaces and utensils used both domestically and in the food industry. Particularly, the biofilms associated with the surfaces used in the food industry, pose a problem of great relevance because said biofilms can become in contact with food, and can cause diseases in the consumer once ingested said contaminated food. In addition, said biofilms can cause significant economic losses because contaminated food is discarded to avoid diseases associated with its consumption.
    [0016]
    [0017] On the other hand, it is important to take into account the problems associated with increased antimicrobial resistance and cross resistance between antibiotics (used in therapy) and biocides (used as disinfectants). Disinfectant formulas should be based on components that do not create long-term resistance, as this would make therapeutic treatments for pathogen infections that are increasingly resistant and invincible more difficult. Currently, the problem of antibiotic resistance is one of the most alarming in the European Community due to deaths caused by multidrug-resistant bacteria (approximately 25,000 people per year in Europe) and also due to the high cost of therapeutic treatments.
    [0018]
    [0019] In the state of the art, much information has been described regarding various compositions that allow biofilms to be eliminated. However, the disinfectant compositions present in the state of the art usually contain among their surfactant and detergent components that are toxic and pollutants to the environment.
    [0020]
    [0021] Therefore, it is considered essential to develop a composition capable of effectively eliminating and / or preventing the formation of biofilms of pathogenic microorganisms, but that at the same time is a composition that does not include toxic bactericidal agents or pollutants for the environment, and that it also does not include components responsible for generating microbial resistance. The achievement of this composition would help the effective elimination of these biofilm-forming pathogens on the different surfaces, interrupting, for example, the process of dissemination and transmission of infectious agents to both healthcare personnel and patients in the clinical setting and contamination. of food in the food industry.
    [0022] DESCRIPTION OF THE INVENTION
    [0023]
    [0024] Brief Description of the Invention
    [0025]
    [0026] In the present invention, the effect of a disinfectant composition comprising lactic acid, hydrogen peroxide and EDTA (hereinafter composition of the invention) on biofilms composed of Gram-positive or Gram-negative bacteria, and also on mixed biofilms formed by multiple species. These biofilms are very resistant to the different treatments applied due to the low diffusion of antimicrobial agents in the matrix of exopolysaccharides produced by the same microorganisms. The composition of the invention allows avoiding the development of these biofilms and also eliminating them once established on different surfaces. The composition of the invention allows the solubilization of the biofilm structure and the diffusion of antimicrobial agents that act synergistically to destroy the microorganisms that make up said structure.
    [0027]
    [0028] The composition of the invention turns out to be very effective in removing biofilms and is characterized by not containing toxic bactericidal agents or pollutants from the environment. The composition of the invention comprises natural components produced by lactic acid bacteria, bacteria with the GRAS status "Generally Recognized As Safe", with antimicrobial effect such as hydrogen peroxide and lactic acid, which qualifies it as a natural product with antimicrobial effects On the other hand, the components included in the composition of the invention do not have a specific target and therefore no bacterial resistance is created after successive applications as it does with quaternary ammonium compounds (cetylpyridinium chloride for example) It is important to indicate that hydrogen peroxide is rapidly degraded to oxygen and water, so this component does not pose a risk to the environment as a pollutant, while lactic acid as an environmentally friendly natural ingredient easily degrades in CO2, CO and CH4.
    [0029]
    [0030] Particularly the composition of the invention comprises:
    [0031]
    [0032] • Two bactericidal agents: lactic acid and hydrogen peroxide. Lactic acid is an antimicrobial agent with a broad spectrum of action that is used in the food industry for its acidifying power rather than antimicrobial. In As for hydrogen peroxide, it is a potent oxidizing agent and a bactericidal agent that allows, in the first place, to weaken the integrity of the biofilm in order to exert its bactericidal action on the microorganisms embedded in said structure. Both antimicrobials act synergistically to eliminate all microorganisms.
    [0033] • EDTA as a chelating agent and inhibitor of efflux pumps. EDTA acts with the objective of extracting membrane ions from both Gram-negative and Gram-positive bacteria and thus facilitate the bactericidal action of the bactericidal agents used. It also acts as an inhibitor of antimicrobial efflux pumps (responsible for nonspecific resistance) and thus increases the susceptibility of bacteria to the bactericidal action of antimicrobials.
    [0034]
    [0035] The three components act synergistically at precise concentrations to eliminate all microorganisms including sporulated bacteria such as Bacillus cereus whose elimination is a challenge for the food industry due to the resistance of its spores to both physical and chemical treatments.
    [0036]
    [0037] The contact time with highly contaminated surfaces (107-108 UCF / ml) should be at least 2 minutes, preferably between 5-15 minutes, to reduce more than 99% of the biofilm's microbial population. Lactic acid and hydrogen peroxide are among the antimicrobial substances produced by lactic acid bacteria (BAL). Lactic acid, as the sole or predominant fermentation product in LABs, plays a crucial role in food preservation, where it is produced up to 8% in the fermentation process. The antimicrobial effect of lactic acid is able to inhibit bacterial growth through disruptive action in the cytoplasmic membrane that leads to loss of proton motive force and filtration of intracellular ions and constituents of Gram-positive and Gram-negative bacteria . In addition, from the point of view of safety, lactic acid is not considered to be associated with chronic health risks and does not represent any risk to the environment.
    [0038]
    [0039] Thus, the composition of the invention allows three important problems associated with surface disinfection to be solved: i) it is capable of effectively removing biofilms in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) no Contains toxic bactericidal agents used in most agents disinfectants due to their destabilizing power of biofilm integrity but at the same time they are associated with a high degree of toxicity and environmental pollution and iii) it is able to inhibit efflux pumps responsible for the resistance of bacteria to agents antimicrobials Having only antimicrobial agents with non-specific targets, it does not create resistance after repeated applications such as for example with quaternary ammonium biocides that are used in different formulations such as cetylpyridinium.
    [0040]
    [0041] Therefore, the composition of the invention can be applied for the elimination of biofilms comprised of pathogenic microorganisms in the health sector, the food industry and even the home. In the food industry sector, the composition of the invention can be used to decontaminate surfaces where food is handled, produced or packaged. In addition, machinery or instruments used in these environments can be disinfected. In this sense, companies in the food industry, slaughterhouses and even food sales areas may have an interest in this type of products, especially when it is a product that lacks toxicity or danger to create microbial resistance. In the health or clinical sector, its use also allows decontamination of surfaces and instruments to prevent the development of biofilms or eliminate them once established. Therefore, contagion and nosocomial infections that may arise in this environment would be avoided. At the domestic level, it is important to note that the composition of the invention offers a high disinfectant power being a natural product, which means that it lacks toxicity. The composition of the invention can also be used for the treatment of patients suffering from bacterial infections and / or poisonings caused by heavy metals.
    [0042]
    [0043] Therefore, the first aspect of the present invention refers to a composition comprising hydrogen peroxide, lactic acid and EDTA, characterized by not comprising toxic bactericidal agents. In a preferred aspect, the composition of the invention is characterized by not comprising phenols, heavy metals, aldehydes and detergents. In a preferred aspect, the composition of the invention is characterized by not comprising: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, Sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate. In a preferred aspect, the composition of the invention comprises: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM of EDTA In a preferred aspect, the composition of the invention comprises: 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
    [0044]
    [0045] The second aspect of the present invention refers to a composition comprising bactericidal agents consisting exclusively of hydrogen peroxide and lactic acid, and an efflux pump inhibitor chelating agent for example EDTA. In a preferred aspect, the composition of the invention comprises two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as an efflux pump inhibiting chelating agent at least 12.5 - 25 mM EDTA. In a preferred aspect, the composition of the invention comprises two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibiting the 25 mM efflux pump of EDTA.
    [0046]
    [0047] The third aspect of the present invention refers to a composition comprising at least two bactericidal agents consisting of hydrogen peroxide and lactic acid and EDTA as an efflux pump inhibiting chelating agent. In a preferred aspect, the composition of the invention comprises at least two bactericidal agents consisting of at least 3-6% hydrogen peroxide and at least 2.2-4.4% lactic acid and as a chelating agent inhibiting the efflux pump at minus 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises at least two bactericidal agents consisting of 6% hydrogen peroxide and 4.25% lactic acid and as a chelating agent inhibiting the 25 mM efflux pump of EDTA.
    [0048]
    [0049] The fourth aspect of the invention refers to an ex vivo method (outside the human or animal body) for the elimination of biofilms formed by at least one bacterium, on any utensil or surface, which comprises the application of the composition of the invention above. described. In a preferred aspect, the composition of the invention is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes. In a preferred aspect, the bacteria that make up the biofilm, which would be eliminated by the composition of the invention, are selected from the group comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
    [0050] The fifth aspect of the present invention refers to the ex vivo use of the composition of the invention as a disinfectant. In a preferred aspect, the invention refers to the use of the composition of the invention for the removal of biofilms formed by at least one bacterium. In a preferred aspect the composition is applied to the biofilm for at least 2 minutes, preferably between 5 and 15 minutes. In a preferred aspect the bacteria that make up the biofilm are: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
    [0051]
    [0052] The sixth aspect of the present invention refers to the composition of the invention for use as a medicament, particularly in the treatment of patients suffering from bacterial infections and / or poisoning caused by heavy metals. In a preferred aspect, the bacterium is selected from the list comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449. In another preferred aspect, the heavy metal is Cadmium.
    [0053]
    [0054] It is important to note that, in a particularly preferred aspect, the composition of the invention comprises precisely established concentrations or amounts in order to achieve a synergistic effect among its components that results in the achievement of a triple technical effect: i) elimination of the biofilm in very short times of at least 2 minutes, preferably between 5-15 minutes, ii) without generating toxicity and iii) at the same time inhibiting efflux pumps responsible for the resistance of bacteria to antimicrobial agents. The concentrations or amounts precisely established to achieve said synergistic effect are: at least 3-6% hydrogen peroxide, at least 2.2-4.4% lactic acid and at least 12.5-25 mM EDTA. In a preferred aspect, the composition of the invention comprises 6% hydrogen peroxide, 4.25% lactic acid and 25 mM EDTA.
    [0055]
    [0056] For the purposes of the present invention the following terms are defined:
    [0057]
    [0058] • The term "that understands" means that it includes, but is not limited to what follows the word "that understands." Therefore, the use of the term "comprising" indicates that the items listed are mandatory or mandatory, but that other items are optional and may or may not be present.
    [0059]
    [0060] • By "consisting of" is understood to include, and is limited to what follows the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the elements listed are mandatory, and that other elements may not be present.
    [0061]
    [0062] • “Disinfectant” means a product that eliminates or prevents the establishment of microorganisms in general and bacteria in particular. The use of a disinfectant allows to limit or even completely eliminate the contamination caused by microorganisms.
    [0063]
    [0064] • "Toxic bactericidal agent" means any agent capable of causing the death of bacteria but at the same time exhibiting a certain toxicity to humans and animals after exposure to said agent. Examples of toxic bactericidal agents are: phenols, heavy metals, aldehydes and detergents, particularly mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, Sodium lauryl sarcosinate, Sodium dodecyl diphenyl ether disulfonate.
    [0065]
    [0066] Description of the figures
    [0067]
    [0068] Figure 1 Antibacterial activity of the composition of the invention in mono and multispecific biofilms (the cocktail of six bacteria: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and UJ Salmonella 34idis) for 0 min (control), 5 minutes (T5), 10 minutes (T10), 20 minutes (T20) and 30 minutes (T30) at room temperature as determined by the determination of the viable count (Log10 CFU / ml). On the Y axis the Log10 CFU / ml and the X axis are shown.
    [0069]
    [0070] Figure 2 Visualization of the inactivation of the multispecific biofilm using the BacLight Live / Dead (Invitrogen) viability kit after treatment with the composition of the invention (100% v / v) for 0 minutes (A, Control), 2 minutes (B) , 5 minutes (C) and 10 minutes (D) at room temperature and resuspension of the biofilm in PBS. All images were obtained using LEICA TCS SP5 II confocal microscope (objective x63) and zoom 2.5 (A and D), 1.5 (C) and 1 (B).
    [0071] Figure 3. Photos of confocal microscopy of biofilm of the cocktail of bacteria treated or not treated with the compound. Left (control, living cells), center (treatment for 5 minutes, large number of dead cells) and right (treatment for 10 minutes, all dead cells).
    [0072]
    [0073] Figure 4 The composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the bacterial cocktail in TSB allowed the inhibition of the expression of genes encoding the efflux pumps EfrAB (efrA and efrB genes) and NorE (the norE gene). C (Control). T (treated with% MIC of the composition of the invention). Y axis (Normalized relative expression).
    [0074]
    [0075] Detailed description of the invention
    [0076]
    [0077] MATERIAL AND METHODS
    [0078]
    [0079] Example 1. Strains and bacterial growth conditions.
    [0080]
    [0081] In the present invention Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449 were used. The strains were grown in Tryptone Soya Broth (TSB) (Fluka, Madrid, Spain) at 37 ° C for 24 hours. The cultures were kept in 20% glycerol at -20 ° C and -80 ° C for short and long term storage, respectively.
    [0082]
    [0083] Example 2. Effect of the composition of the invention on the growth of planktonic cells.
    [0084]
    [0085] To determine the minimum inhibitory concentration (MIC) and the minimum bactericide concentration (MBC) of the composition of the invention (6% H2O2, 4.25% lactic acid and 25 mM EDTA), the broth microdilution method was used . Overnight, bacterial cultures grown in TSB broth at 37 ° C for 24 hours were diluted 1/10 (v / v) in fresh TSB broth and 20 ^ each well of the 96-well microtiter plates were added. Subsequently, 180 µl of TSB broth supplemented with the composition of the invention was added at different concentrations (0.25-50%, v / v). Plates were incubated at 37 ° C under aerobic conditions for 24 hours and bacterial growth was evaluated by the presence of turbidity. The wells they exhibited The absence of turbidity was subjected to determination of viable counts (CFU / ml) by sowing the samples (10 ^ l) in Soya triptone agar plates (TSA). Subsequently the plates were incubated at 37 ° C for 24 hours. MIC was defined as the lowest concentration of the composition of the invention that inhibits visible growth and MBC was defined as the lowest concentration of the composition of the invention that kills bacteria (99%). Each experiment was done in triplicate.
    [0086]
    [0087] Example 3. Determination of anti-biofilm activity.
    [0088]
    [0089] The anti-adhesion properties of the composition of the invention to different bacterial strains (S. aureus CECT 4468, L. monocytogenes CECT 4032, E. faecalis S-47, B. cereus CECT 5148, E. coli CCUG 47553 and S. Enteritidis UJ3449) and the cocktail of all strains were tested in microtiter plates. Overnight, bacterial cultures grown in TSB broth at 37 ° C for 24 hours were diluted 1/10 (v / v) in fresh TSB broth and 20 ^ each well of the 96-well microtiter plates were added. Subsequently, the wells were completed with 180 µl of TSB broth supplemented with the composition of the invention at sub-MIC concentrations (1/2 MIC of the composition of the invention for each strain up to 20% of the composition of the invention, v / v). Controls without the composition of the invention consisted of 180 µl of TSB broth. The plates were incubated at 37 ° C under aerobic conditions for 24 hours and the wells were washed with phosphate buffered saline (PBS). The anti-biofilm activity of the composition of the invention was determined by staining the washed wells with 100 µl of 1% violet glass (w / v) that were incubated at room temperature for 15 minutes. Subsequently, the absorbance at 590 nm was determined using a microplate reader (iMark microplate absorbance reader, Bio-Rad instrument). The percent inhibition of biofilm formation was determined using the following formula as described by Zmantar et al. (2017):
    [0090]
    [0091]
    [0092]
    [0093]
    [0094] Example 4. Antimicrobial effect of the composition of the invention on the preformed biofilm.
    [0095] An inoculum was used with 2% of each bacterium and the cocktail of all strains in TSB for the preparation of the biofilm that was grown in 96-well microtiter plates for 24 hours 37 ° C. After incubation, the culture broth containing non-adherent bacteria was removed and the wells were washed with sterile PBS. The biofilms were treated with the composition of the invention for different periods of time (5, 10, 15, 20 and 30 minutes) at room temperature. After the treatments, the composition of the invention was removed and the wells were incubated with 200 µl of D / E neutralizing broth (Difco, Barcelona) for 5 minutes at room temperature and then washed with 200 µl of PBS. The biofilms were resuspended in 200 µl of PBS and then seeded in TSA. The plates were incubated at 37 ° C for 24 hours to determine the total number of CFU / ml.
    [0096]
    [0097] Example 5. Microscopic evaluation of the effect of the composition of the invention on the biofilm.
    [0098]
    [0099] Imaging of the biofilm treated with the composition of the invention was performed using LIVE / DEAD BacLight ™ (Thermo Fisher Scientific, Waltham, MA, USA) and a confocal laser scanning microscope (LEICA TCS-SP5 II, Mannheim , Germany) equipped with Plan-Apochromat 63x / 1.4 objective. After culturing the biofilm in a microtiter plate (200 µl) as described above, some wells were not treated with the composition of the invention (control) and the others were subjected to treatment with the composition of the invention for 5 and 10 minutes at room temperature, they were washed with sterile PBS and resuspended in 50 µl of PBS. Subsequently, 20 ^ l of the suspensions (control and treatment with the composition of the invention) were completed with 0.5 ^ LIVE / DEAD staining and subsequently placed on a glass slide and images were obtained using a scanning microscope confocal laser.
    [0100]
    [0101] Example 6. Effect of the sub-inhibitory concentration of the composition of the invention on biofilm resistance.
    [0102]
    [0103] 1/2 MIC of the composition of the invention in TSB broth (2 ml) was added to the cocktail of the six bacterial strains (2%) and subsequently incubated for 18 hours at 37 ° C in tubes and also in tubes. 12-well microtiter plates for biofilm formation. RNA extraction was performed from planktonic bacterial cultures and also of multispecies biofilms using the Direct-zol ™ RNA kit (Zymo Research, USA) according to the manufacturer's instructions.
    [0104]
    [0105] Example 7. Statistical analysis.
    [0106]
    [0107] All analyzes were performed in triplicate. Statistical analyzes were performed using the Excel 2007 program (Microsoft Corporation, Redmond, Washington, USA). To determine the averages and standard deviations. Statistical evaluation of biofilm development inhibition was performed by analysis of variance (ANOVA) using Statgraphics Centurion XVI software (Statpoint Technologie, Warrenton, Virginia, USA). The same software was used to perform the Shapiro-Wilk and Levene tests to verify the normality of the data and perform the bilateral Tukey multiple contrast to determine the pairwise differences between the strains, where the level of significance was established in the value P of <0.05.
    [0108]
    [0109] RESULTS
    [0110]
    [0111] Example 8. Antimicrobial activity of the composition of the invention in planktonic cultures.
    [0112]
    [0113] Table 1 shows the antimicrobial effect that the composition of the invention exerts on the growth of the planktonic cells of each bacterium and also on the cocktail of all the bacteria tested. Table 1 shows the determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the composition of the invention.
    [0114]
    [0115] Table 1
    [0116]
    [0117]
    [0118] High susceptibility of the planktonic bacterial strains was detected since they exhibited very low MICs ranging from 0.15% to 0.4% of the composition of the invention (v / v) with L. monocytogenes CECT 4032 and E. faecalis S-47 strains more susceptible However, S. aureus CECT 4468 was less susceptible than other species and also the cocktail. As for the MBC values, they were very similar, ranging between 0.2% and 0.5% of the composition of the invention (v / v). In this sense, the composition of the invention was effective against Gram-positive and Gram-negative bacteria and also against the cocktail made with the six bacteria since it was also eliminated in view of the low concentration of the composition of the invention (0.5 %).
    [0119]
    [0120] Example 9. Inhibition of biofilm formation by the composition of the invention.
    [0121]
    [0122] A strong inhibition of biofilm development was achieved using the composition of the invention for each strain and for the bacterial cocktail. The results obtained showed that 80-91% of the inhibition of the developing biofilm was achieved for individual strains and also the cocktail ( Table 2 ). In addition, the use of 1/2 MIC of the composition of the invention allowed a 33-50% inhibition of biofilm development ( Table 2 ). These results indicated that the composition of the invention inhibited bacterial adhesion to polystyrene ( Table 2 ).
    [0123]
    [0124] Table 2
    [0125]
    [0126]
    [0127]
    [0128] ± DS, standard deviation of three independent experiments.
    [0129] * Each different lowercase letter represents significant differences according to Tukey's HSD between strains (p <0.05).
    [0130] Example 10. Evaluation of the antimicrobial effect of the composition of the invention in biofilms.
    [0131]
    [0132] The preformed biofilms of different bacterial strains in microtiter plates were subjected to the antimicrobial effect of the composition of the invention for several contact times (5, 10, 15, 20 and 30 minutes). The results obtained showed the total bactericidal effect of the composition of the invention against all bacterial strains and also the cocktail after all contact times varying from 5 to 30 minutes (see Figure 1 ). In addition, confocal microscopy revealed that the composition of the invention had a strong effect on the multi-species biofilm since no viable cells of the biofilms treated with the composition of the invention (5 and 10 minutes) were detected within the biofilm matrix (see Figure 2 BD ), compared to controls not treated with the composition of the invention (see Figure 2 A ). However, dead cells were visualized (see Figure 2 C and D ).
    [0133]
    [0134] Example 11. The composition of the invention inhibits efflux pumps.
    [0135]
    [0136] The composition of the invention is capable of inhibiting efflux pumps that act as mechanisms of nonspecific resistance in bacteria (particularly EfrAB and NorE efflux pumps). This is demonstrated in the present invention by analyzing the expression of said genes in the cocktail of the bacteria studied in the absence and presence of low concentrations of the compound (1/2 MIC). The composition of the invention added to a sub-inhibitory concentration (1/2 MIC) to the bacterial cocktail in TSB allowed the inhibition of the expression of genes encoding the efflux pumps EfrAB (efrA and efrB genes) and NorE (the norE gene) as shown in Figure 4 . This inhibition of efflux pumps reduces the spread of pathogens resistant to different antimicrobials. Thus, the development of biofilms with greater resistance to antimicrobials is avoided. In Figure 4 it is clearly seen that the application of the composition of the invention at a sub-inhibitory growth concentration (1/2 MIC) has a low expression of the coding genes of the EfrAB and NorE efflux pumps, which play an important role in intrinsic and nonspecific resistance to different antimicrobial agents (antibiotics and biocides). Therefore, on the one hand, thanks to the inhibitory action of efflux pumps, the composition of the invention allows to enhance the antimicrobial action of the two bactericidal agents (lactic acid and hydrogen peroxide) by blocking one of the defense mechanisms bacterial, thus allowing to restore the antimicrobial activity of various Antimicrobial compounds that have lost their activity due to the presence of efflux pumps. On the other hand, the use of the composition of the invention, by blocking or inhibiting efflux pumps, will reduce the existing emergency regarding the existence of multimicrobial resistant bacteria. Currently, efflux pumps represent very attractive targets to reduce the spread of antimicrobial resistance (antibiotics and biocides). Particularly, the mechanism by which EDTA is believed to promote the inhibition of the efflux pump is by sequestering calcium ions, thus, hydrolysis of ATP cannot occur and the export pumps cannot be put into operation. The ABC conveyor family, as is the case with the EfrAB export pump.
    权利要求:
    Claims (15)
    [1]
    1. Composition comprising hydrogen peroxide, lactic acid and EDTA, characterized by not comprising toxic bactericidal agents.
    [2]
    2. Composition according to claim 1, characterized in that the toxic bactericidal agent is selected from the list comprising: phenols, heavy metals, aldehydes and detergents.
    [3]
    3. Composition according to any of the preceding claims, characterized in that the toxic bactericidal agent is selected from the list comprising: mandelic acid, peracetic acid, SDS, cetylpyridinium chloride, Sodium Cocoyl Sarcosinate, Sodium lauryl sarcosinate and / or Sodium dodecyl diphenyl ether disulfonate.
    [4]
    4. Composition according to any of the preceding claims, comprising:
    to. At least 3-6% hydrogen peroxide.
    b. At least 2.2-4.4% lactic acid.
    c. 12.5-25 mM EDTA.
    [5]
    5. Composition according to any of the preceding claims, comprising:
    to. 6% hydrogen peroxide.
    b. 4.25% lactic acid.
    c. 25 mM EDTA.
    [6]
    6. Method for the removal of biofilms formed by at least one bacterium, present on any surface or utensil, comprising the application of the composition of claims 1 to 5.
    [7]
    7. Method according to claim 6, wherein the composition is applied to the biofilm for at least 2 minutes, preferably for 5-15 minutes.
    [8]
    8. Method according to claims 6 or 7, wherein the bacteria that make up the biofilm are: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
    [9]
    9. Use of the composition of claims 1 to 5 as a disinfectant of any type of utensil or surface.
    [10]
    10. Use according to claim 9 for the removal of biofilms formed by at least one bacterium.
    [11]
    11. Use according to claims 9 or 10, wherein the composition is applied to the biofilm for at least 2 minutes, preferably for 5-15 minutes.
    [12]
    12. Use according to claims 9 to 11, wherein the bacteria that make up the biofilm are: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
    [13]
    13. Composition according to any one of claims 1 to 5, for use in the treatment of patients suffering from bacterial infections and / or poisoning caused by heavy metals.
    [14]
    14. Composition for use according to claim 13, wherein the bacterium is selected from the list comprising: Staphylococcus aureus CECT 4468, Listeria monocytogenes CECT 4032, Enterococcus faecalis S-47, Bacillus cereus CECT 5148, Escherichia coli CCUG 47553 and Salmonella Enteritidis UJ3449.
    [15]
    15. Composition for use according to claim 13, wherein the heavy metal is Cadmium.
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    同族专利:
    公开号 | 公开日
    WO2019122470A1|2019-06-27|
    ES2717798B2|2020-09-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1993002973A1|1991-08-05|1993-02-18|Trawöger, Werner|Anti-fouling agent for wet surfaces|
    US5731275A|1994-04-05|1998-03-24|Universite De Montreal|Synergistic detergent and disinfectant combinations for decontaminating biofilm-coated surfaces|
    US20020016278A1|1998-11-06|2002-02-07|Jean Barbeau|Bactericidal and non-bactericidal solutions for removing biofilms.|
    US20050153031A1|2004-01-09|2005-07-14|Ecolab Inc.|Methods for washing carcasses, meat, or meat products with medium chain peroxycarboxylic acid compositions|
    US20090312279A1|2005-12-23|2009-12-17|Sterilex Technologies, Llc|Antimicrobial compositions|
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    PCT/ES2018/070785| WO2019122470A1|2017-12-22|2018-12-05|Disinfectant composition|
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